The field of the invention is cellular proteins which mediate entry of viruses into cells.
Herpesviruses are ubiquitous viruses which are the causative agents of numerous diseases in both humans and animals. These viruses are enveloped double stranded icosahedral DNA containing viruses, which envelope is acquired by budding of the nucleocapsid through the inner nuclear membrane. Members of the herpesvirus family that are important human pathogens include herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella zoster virus (VZV), Epstein Barr virus (EBV), cytomegalovirus (CMV), and human herpesviruses type 6, type 7 and type 8 (HHV-6, HHV-7 and HHV-8).
A key feature of viral infection of cells is the binding of one or more viral proteins with one or more cellular proteins expressed on the surface of a cell, which binding results in entry of the virus into the cell. Members of the alphaherpesvinrs subfamily of the herpesviruses typically have a broad host range and a short replicative cycle, are highly cytotoxic to cultured cells, and can establish latent infections in cells of the nervous system of the natural host (Roizman, 1993, The family herpesviridae. In: The Human Herpesviruses (Roizman, Whitley and Lopez, Eds.), pp. 1-9. Raven Press Ltd., New York). Human and animal representatives of the alphaherpesvirus subfamily exhibit common requirements for entry into cells (Mettenleiter, 1995, Molecular properties of alphaherpesviruses used in transneuronal pathway tracing. In: Viral Vectors, pp. 367-393. Academic Press, Inc; Spear, 1993, Sem. Virol. 4:167-180). In most cases, five viral envelope glycoproteins (gB, gC, gD, gH and gL) mediate virus binding to cells and entry therein. The initial interaction of virus with cells is binding of gC, and in some cases also gB, to cell surface glycosaminoglycans (GAGs), and preferentially to heparan sulfate. Although gC is dispensable for the infection of many cultured cells, gB, gD, gH arid gL are required for mediating the fusion between the virion envelope and the cell membrane that allows viral penetration. Various lines of evidence point to the interaction of some of these envelope glycoproteins, particularly gD, with cell surface receptors other than heparan sulfate and to competition among alphaherpesviruses for binding to gD receptors (Campadelli-Fiume et al., 1988, J. Virol. 62:159-167; Chase et al., 1993, Virology 194:365-369; Johnson et al., 1990, J. Virol. 64:2569-2576; Johnson and Ligas, 1988, J. Virol. 62:4605-4612; Karger and Mettenleiter, 1993, Virology 194:654-664; Lee and Fuller, 1993, J. Virol. 67:5088-5097; Liang et al., 1991, J. Virol. 65:1124-1132; Petrovskis et al., 1988, J. Virol. 62:2196-2199).
An expression cloning assay has been devised for isolating plasmids encoding cell surface proteins that can mediate herpes simplex virus type 1 (HSV-1) entry (Montgomery et al., 1996, Cell 87:427-436). This assay relies on the use of Chinese hamster ovary (CHO) cells, which express GAGs required for virus binding to cells but are resistant to the entry of certain HSV-1 strains such as HSV-1 (KOS) (Shieh et al., 1992, J. Cell Biol. 116:1273-1281). Expression libraries, or sub-divisions of the libraries, that contain plasmids capable of conferring susceptibility to HSV-1(KOS) can be identified by transfecting the CHO cells and then assaying for infection with a recombinant HSV-1(KOS) expressing a reporter gene.
A cell surface protein having herpesvirus entry activity was identified using the aforementioned assay. This cell surface protein is a previously undescribed member of the human TNF receptor family, which was originally named herpesvirus entry mediator (HVEM) and is designated herein as herpesvirus entry protein A (HveA). HveA is disclosed in U.S. application Ser. No. 08/509,024, filed on Jul. 28, 1995, which is hereby incorporated herein by reference in its entirety. HveA is a type I membrane glycoprotein with cysteine-rich repeats in the ectodomain that are characteristic of the TNF receptor family (Montgomery et al., 1996, Cell 87 :427) and with a cytoplasmic domain that can interact with members of the TRAF family of signaling molecules (Hsu et al., 1997, J. Biol. Chem. 272:13471-13474; Marsters et al., 1997, J. Biol. Chem. 272:14029-14032). HveA is a receptor for two members of the TNF family, lymphotoxin a and LIGHT (Mauri et al., 1998, Immunity 8:21-30). HveA also binds to isolated HSV-1 or HSV-2 gD and to gD in virions (Nicola et al., 1998, J. Virol. 72:3595-3601; Whitbeck et al., 1997, J. Virol. 71:6083-6093).
HveA is expressed in many fetal and adult human tissues, including lung, liver and kidney (Montgomery et al., 1996, Cell 87:427), but appears to be most abundantly expressed in lymphoid organs and cells (Hsu et al., 1997, J. Biol. Chem. 272:13471-13474; Kwon et al., 1997, J. Biol. Chem. 272:14272-14276; Marsters et al., 1997, J. Biol. Chem. 272:14029-14032). The use of anti-HveA antibodies that blocked HSV entry established that HveA serves as the principal co-receptor for entry of HSV-1(KOS) into activated human T lymphocytes (Montgomery et al., 1996, Cell 87:427). However, the antibodies did not protect a number of other human cell types from infection, indicating that there are other co-receptors for HSV entry.
Another indication for the existence of multiple independent co-receptors for HSV entry was the finding that, although HveA expression in CHO cells enhanced the entry of all wild-type HSV-1 and HSV-2 strains tested, HveA failed to mediate the entry of three mutant HSV-1strains (Montgomery et al., 1996, Cell 87:427). Two of these strains, designated HSV-1(KOS)Rid1 and HSV-1(KOS)Rid2 and abbreviated herein as KOS-Rid1 and KOS-Rid2, are viable mutants which were selected for theft ability to overcome interference with viral entry imposed by the expression of wild-type HSV-1 gD in cells (Dean et al., 1994, Virology 199:67-80). The third strain, HSV-1(ANG), was isolated from a clinical specimen (Munk and Donner, 1963, Arch. Gesamte Virus-forsch. 13:529-540) under conditions that would be expected to inhibit the replication of wild-type HSV (under an overlay of agar containing inhibitory sulfated polysaccharides) and perhaps select for viral variants. This strain proved to be as resistant to gD-mediated interference as KOS-Rid1 and KOS-Rid2 (Dean et al., 1994, Virology 199, 67-80). All three strains have amino acid substitutions, in gD at position 27 (Q27P or Q27R), that are sufficient to confer the mutant phenotype of resistance to gD-mediated interference. Consistent with the failure of the mutant strains to use HveA for entry, the mutant forms of gD failed to bind HveA whereas wild-type forms of HSV-1 and HSV-2 gD were able to bind HveA (Whitbeck et al., 1997, J. Virol. 71 :6083-6093). Because the mutant HSV-1 strains can infect a number of human or other cell types, despite failure to use HveA for entry, other co-receptors for entry must be expressed in these cells.
Currently, there are no fully effective treatments for herpesvirus infection of humans, which infections are of ten severe, and sometimes even fatal. Clearly, the identification of compounds which inhibit entry of herpesvirus into cells would greatly facilitate treatment of herpesviruses in humans. To date, there are no commercially available compounds which are approved for treatment of herpesvirus infection, which compounds inhibit entry of virus into cells. Thus, there is a long felt need for the identification of and methods of use of such compounds. The present invention satisfies this need.
The invention relates to a cellular herpesvirus entry protein, or a mutant, a homolog, a derivative, a variant or a biologically active fragment thereof, suspended in a pharmaceutically active carrier in an amount effective to inhibit entry of an alphaherpesvirus into a cell, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily.
In one aspect, the cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC.
In another aspect, the alphaherpesvirus is selected from the group consisting of HSV-1, HSV-2, and the animal viruses, pseudorabies virus (PRV) and bovine herpesvirus 1 (BHV-1).
The invention also relates to a recombinant cell comprising an isolated nucleic acid encoding a cellular herpesvirus entry protein, or a mutant, cL homolog, a derivative, a variant or a biologically active fragment thereof, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily and the herpesvirus is an alphaherpesvirus.
In one aspect, cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC.
In another aspect, the alphaherpesvirus is selected from the group consisting of HSV-1, HSV-2, PRV and BHV-1.
In yet another aspect, the cell is selected from the group consisting of Chinese hamster ovary cells, murine melanoma cells, and swine testes cells.
Also included in the invention is a vector comprising an isolated nucleic acid encoding a cellular herpesvirus entry protein, or a mutant, a homolog, a derivative, a variant or a biologically active fragment thereof, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily and the herpesvirus is an alphaherpesvirus.
In one aspect, the cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC.
In another aspect, the alphaherpesvirus is selected from the group consisting of HSV-1, HSV-2, PRV and BHV-1.
The invention further relates to an anti-cellular herpesvirus protein compound, wherein the compound binds herpesvirus glycoprotein D.
In one aspect, the cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC.
In another aspect, the herpesvirus is selected from the group consisting of HSV-1, HSV-2, PRV and BHV-1.
The invention additionally relates to an anti-cellular herpesvirus protein compound, wherein the compound is selected from the group consisting of an antisense oligonucleotide, an antibody specific for the cellular herpesvirus protein, a peptide and a peptidomimetic.
Also included in the invention is a method of identifying a compound capable of inhibiting entry of an alphaherpesvirus into a cell. The method comprises providing a population of cells which express a cellular herpesvirus entry protein, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily, infecting the cells in the presence or absence of a test compound, and measuring the level of entry of an alphaherpesvirus into the cells, wherein a lower level of entry of the virus into the cells in the presence of the test compound compared with the level of entry of the virus into the cells in the absence of the test compound is an indication that the test compound is an anti-cellular herpesvirus entry protein compound.
The invention also includes an anti-cellular herpesvirus entry protein compound identified by the method of identifying a compound capable of inhibiting entry of an alphaherpesvirus into a cell. The method comprises providing a population of cells which express a cellular herpesvirus entry protein, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily, infecting the cells in the presence or absence of a test compound, and measuring the level of entry of an alphaherpesvirus into the cells, wherein a lower level of entry of the virus into the cells in the presence of the test compound compared with the level of entry of the virus into the cells in the absence of the test compound is an indication that the test compound is an anti-cellular herpesvirus entry protein compound.
The invention also relates to a method of inhibiting entry of an alphaherpesvirus into a cell comprising adding to the cell an anti-cellular herpesvirus entry protein compound thereby inhibiting entry of the virus into the cell.
In addition, the invention relates to a method of treating an alphaherpesvirus infection in an animal comprising administering to the animal an anticellular herpesvirus entry protein compound, wherein the cellular herpesvirus entry protein is a member of the immunoglobulin superfamily.
In one aspect, the animal is a human.
In another aspect, the herpesvirus is selected from the group consisting of HSV-1 and HSV-2.
The invention further relates to a composition comprising a soluble alphaherpesvirus glycoprotein D-binding cellular herpesvirus entry protein.
In one aspect, the cellular herpesvirus entry protein is selected from the group consisting of HveB and HveC. In a preferred embodiment, the .HveB and the HveC are truncated.